技术资料

Human pluripotent stem cells (HPSCs) cultured in conditions that maintain pluripotency via FGF and TGFβ signaling have been described as being in a primed state. These cells have been shown to exhibit characteristics more closely related to mouse epiblast-derived stem cells than to so called naïve mouse PSCs said to possess a more ground state pluripotency that mimics the early mouse embryo inner cell mass. Initial attempts to create culture conditions favorable for generation of naïve HPSCs from primed HPSCs has required the use of mouse embryonic fibroblasts as a feeder layer to support this transition. A protocol for the routine derivation and maintenance of naïve HPSCs in completely defined conditions is highly desirable for stem cell researchers to enhance the study and clinical translation of naïve HPSCs. Here we describe a standard protocol for transitioning primed HPSCs to a naïve state using commercial RSet media and xeno-free recombinant vitronectin. View Publication

Natural killer (NK) cells are cytotoxic immune cells that can eliminate target cells without prior stimulation. Human induced pluripotent stem cells (iPSCs) provide a robust source of NK cells for safe and effective cell-based immunotherapy against aggressive cancers. In this in vitro study,a feeder-free iPSC differentiation was performed to obtain iPSC-NK cells,and distinct maturational stages of iPSC-NK were characterized. Mature cells of CD56bright CD16bright phenotype showed upregulation of CD56,CD16,and NK cell activation markers NKG2D and NKp46 upon IL-15 exposure,while exposure to aggressive atypical teratoid/rhabdoid tumor (ATRT) cell lines enhanced NKG2D and NKp46 expression. Malignant cell exposure also increased CD107a degranulation markers and stimulated IFN-? secretion in activated NK cells. CD56bright CD16bright iPSC-NK cells showed a ratio-dependent killing of ATRT cells,and the percentage lysis of CHLA-05-ATRT was higher than that of CHLA-02-ATRT. The iPSC-NK cells were also cytotoxic against other brain,kidney,and lung cancer cell lines. Further NK maturation yielded CD56?ve CD16bright cells,which lacked activation markers even after exposure to interleukins or ATRT cells - indicating diminished cytotoxicity. Generation and characterization of different NK phenotypes from iPSCs,coupled with their promising anti-tumor activity against ATRT in vitro,offer valuable insights into potential immunotherapeutic strategies for brain tumors. Graphical abstractImage 1 Highlights•Natural killer (NK) cells were derived from human induced pluripotent stem cells (iPSCs) in the absence of feeder cells.•Various maturational subtypes of iPSC-NK cells were characterized,and the phenotypic and functional properties were studied.•iPSC-NK cells of CD56bright CD16bright phenotype expressed activation markers in response to interleukin stimuli.•iPSC-NK cells were cytotoxic toward human atypical teratoid and rhabdoid tumor (ATRT) cells and other human cancer cells.•The cytotoxicity of iPSC-NK cells against various cancer cells in vitro might be translated into an in vivo immunotherapy. View Publication

Previous studies have shown that maintenance of undifferentiated human embryonic stem (hES) cells requires culture on mouse embryonic fibroblast (MEF) feeders. Here we demonstrate a successful feeder-free hES culture system in which undifferentiated cells can be maintained for at least 130 population doublings. In this system,hES cells are cultured on Matrigel or laminin in medium conditioned by MEF. The hES cells maintained on feeders or off feeders express integrin alpha6 and beta1,which may form a laminin-specific receptor. The hES cell populations in feeder-free conditions maintained a normal karyotype,stable proliferation rate,and high telomerase activity. Similar to cells cultured on feeders,hES cells maintained under feeder-free conditions expressed OCT-4,hTERT,alkaline phosphatase,and surface markers including SSEA-4,Tra 1-60,and Tra 1-81. In addition,hES cells maintained without direct feeder contact formed teratomas in SCID/beige mice and differentiated in vitro into cells from all three germ layers. Thus,the cells retain fundamental characteristics of hES cells in this culture system and are suitable for scaleup production. View Publication

Feeder-independent culture of human embryonic stem cells. View Publication

The continued success of pluripotent stem cell research is ultimately dependent on access to reliable and defined reagents for the consistent culture and cryopreservation of undifferentiated,pluripotent cells. The development of defined and feeder-independent culture media has provided a platform for greater reproducibility and standardization in this field. Here we provide detailed protocols for the use of mTeSR™1 and TeSR™2 with various cell culture matrices as well as defined cryopreservation protocols for human embryonic and human induced pluripotent stem cells. View Publication

STUDY HYPOTHESIS Does a preferential X chromosome inactivation (XCI) pattern exist in female human pluripotent stem cells (hPSCs) and does the pattern change during long-term culture or upon differentiation? STUDY FINDING We identified two independent phenomena that lead to aberrant XCI patterns in female hPSC: a rapid loss of histone H3 lysine 27 trimethylation (H3K27me3) and long non-coding X-inactive specific transcript (XIST) expression during culture,often accompanied by erosion of XCI-specific methylation,and a frequent loss of random XCI in the cultures. WHAT IS KNOWN ALREADY Variable XCI patterns have been reported in female hPSC,not only between different hPSC lines,but also between sub-passages of the same cell line,however the reasons for this variability remain unknown. Moreover,while non-random XCI-linked DNA methylation patterns have been previously reported,their origin and extent have not been investigated. STUDY DESIGN,SAMPLES/MATERIALS,METHODS We investigated the XCI patterns in 23 human pluripotent stem cell (hPSC) lines,during long-term culture and after differentiation,by gene expression analysis,histone modification assessment and study of DNA methylation. The presence and location of H3K27me3 was studied by immunofluorescence,XIST expression by real-time PCR,and mono- or bi-allelic expression of X-linked genes was studied by sequencing of cDNA. XCI-specific DNA methylation was analysed using methylation-sensitive restriction and PCR,and more in depth by massive parallel bisulphite sequencing. MAIN RESULTS AND THE ROLE OF CHANCE All hPSC lines showed XCI,but we found a rapid loss of XCI marks during the early stages of in vitro culture. While this loss of XCI marks was accompanied in several cases by an extensive erosion of XCI-specific methylation,it did not result in X chromosome reactivation. Moreover,lines without strong erosion of methylation frequently displayed non-random DNA methylation,which occurred independently from the loss of XCI marks. This bias in X chromosome DNA methylation did not appear as a passenger event driven by clonal culture take-over of chromosome abnormalities and was independent of the parental origin of the X chromosome. Therefore,we suggest that a culture advantage conferred by alleles on the X chromosome or by XCI-related mechanisms may be at the basis of this phenomenon. Finally,differentiated populations inherited the aberrant XCI patterns from the undifferentiated cells they were derived from. LIMITATIONS,REASONS FOR CAUTION All hPSC lines in this study were cultured in highly similar conditions. Our results may therefore be specific for these conditions and alternative culture conditions might lead to different findings. Our findings are only a first step towards elucidating the molecular events leading to the phenomena we observed. WIDER IMPLICATIONS OF THE FINDINGS Our results highlight the significant extent of aberrant XCI in female hPSC. The fact that these aberrations are inherited by the differentiated progeny may have a significant impact on downstream research and clinical uses of hPSC. In order to achieve the full potential of hPSC,more insight into the XCI status and its stability in hPSC and its effect on the properties of the differentiated progeny is needed. LARGE SCALE DATA Not applicable. STUDY FUNDING AND COMPETING INTERESTS Our research is supported by grants from the Research Foundation - Flanders (FWO-Vlaanderen,grant 1502512N),Generalitat de Catalunya (2014SGR-005214) and the Methusalem grant of the Research Council of the Vrije Universiteit Brussel,on name of K.S. L.V.H. is funded by EMBO (ALTF 701-2013). The authors declare no potential conflict of interest. View Publication

To propose candidates for the prevention or treatment of osteoporosis,we have screened compounds naturally in food for their ability to regulate the differentiation and function of osteoclasts. One of the major green tea flavonoids,(-)-epigallocatechin-3-gallate (EGCG),was found to induce apoptotic cell death of osteoclast-like multinucleated cells after 24 h treatment in a dose-dependent manner (25-100 microM),whereas osteoblasts were not affected. In the present study,we report for the first time a novel cell-death-inducing mechanism triggered by EGCG. The induction of apoptosis by EGCG was suppressed by pretreatment of catalase or calcitonin. It was also suppressed by Fe(III) and Fe(II) chelators. Furthermore,EGCG promoted the reduction of Fe(III) into Fe(II),and the combination of EGCG/Fe(III)/H(2)O(2) induced single-strand DNA breakage in a cell free system. These results indicate that the Fenton reaction is primarily involved in EGCG-induced osteoclastic cell death. View Publication

Tracheal replacement is a promising approach for treating tracheal defects that are caused by conditions such as stenosis,trauma,or tumors. However,slow postoperative epithelial regeneration often leads to complications,such as infection and granulation tissue formation. Ferroptosis,which is an iron-dependent form of regulated cell death,limits the proliferation of tracheal basal cells (TBCs),which are essential for the epithelialization of tissue-engineered tracheas (TETs). This study explored the potential of ferrostatin-1 (FER-1),which is a ferroptosis inhibitor,to increase TBC proliferation and accelerate the epithelialization of 3D-printed TETs. TBCs were isolated from rabbit bronchial mucosal tissues and cultured in vitro. Ferroptosis was induced in TBCs at passage 2,as shown by increased reactive oxygen species (ROS) levels,Fe 2 ⁺ accumulation,decreased ATP contents,and mitochondrial damage. TBCs were treated with FER-1 (1 μM) for 48 h to inhibit ferroptosis. The effects on ROS levels,Fe 2 ⁺ levels,ATP contents,and mitochondrial morphology were measured. For in vivo experiments,FER-1-treated TBCs were seeded onto 3D-printed polycaprolactone (PCL) scaffolds,which were implanted into rabbits with tracheal injury. Epithelial regeneration and granulation tissue formation were evaluated 6 months after surgery. FER-1 treatment significantly reduced ferroptosis marker levels in vitro; that is,FER-1 treatment decreased ROS and Fe 2 ⁺ accumulation,ameliorated mitochondrial structures,and increased ATP levels. TBC proliferation and viability were increased after ferroptosis inhibition. In vivo,the group that received 3D-printed scaffolds seeded with TBCs exhibited accelerated TET epithelialization and reduced granulation tissue formation compared with the control groups. These results suggest that inhibiting ferroptosis with FER-1 improves TBC function,leading to more efficient tracheal repair. Ferrostatin-1 effectively inhibits ferroptosis in tracheal basal cells,promoting their viability and proliferation. This results in faster epithelialization of tissue-engineered tracheas,offering a promising strategy for improving tracheal reconstruction outcomes and reducing complications such as infection and granulation tissue formation. Future studies are needed to further investigate the molecular mechanisms underlying ferroptosis in TBCs and its potential clinical applications. The online version contains supplementary material available at 10.1186/s13287-025-04263-z. View Publication

Fetuses with homozygous alpha-thalassemia usually die at the third trimester of pregnancy or soon after birth. Hence,the disease could potentially be a target for fetal gene therapy. We have previously established a mouse model of alpha-thalassemia. These mice mimic the human alpha-thalassemic conditions and can be used as preclinical models for fetal gene therapy. We tested a lentiviral vector containing the HS 2,3,and 4 of the beta-LCR,a central polypurine tract element,and the beta-globin gene promoter directing either the EGFP or the human alpha-globin gene. We showed that the GFP expression was erythroid-specific and detected in BFU-E colonies and the erythroid progenies of CFU-GEMM. For in utero gene delivery,we did yolk sac vessel injection at midgestation of mouse embryos. The recipient mice were analyzed after birth for human alpha-globin gene expression. In the newborn,human alpha-globin gene expression was detected in the liver,spleen,and peripheral blood. The human alpha-globin gene expression was at the peak at 3-4 months,when it reached 20% in some recipients. However,the expression declined at 7 months. Colony-forming assays in these mice showed low abundance of the transduced human alpha-globin gene in their BFU-E and CFU-GEMM and the lack of its transcript. Thus,lentiviral vectors can be an effective vehicle for delivering the human alpha-globin gene into erythroid cells in utero,but,in the mouse model,delivery at late midgestation could not transduce hematopoietic stem cells adequately to sustain gene expression. View Publication

Autosomal recessive osteopetrosis (ARO) is a group of genetic disorders that involve defects that preclude the normal function of osteoclasts,which differentiate from hematopoietic precursors. In half of human cases,ARO is the result of mutations in the TCIRG1 gene,which codes for a subunit of the vacuolar proton pump that plays a fundamental role in the acidification of the cell-bone interface. Functional mutations of this pump severely impair the resorption of bone mineral. Although postnatal hematopoietic stem cell transplantation can partially rescue the hematological phenotype of ARO,other stigmata of the disease,such as secondary neurological and growth defects,are not reversed. For this reason,ARO is a paradigm for genetic diseases that would benefit from effective prenatal treatment. Using the oc/oc mutant mouse,a murine model whose osteopetrotic phenotype closely recapitulates human TCIRG1-dependent ARO,we report that in utero transplantation of adult bone marrow hematopoietic stem cells can correct the ARO phenotype in a limited number of mice. Here we report that in utero injection of allogeneic fetal liver cells,which include hematopoietic stem cells,into oc/oc mouse fetuses at 13.5 days post coitum produces a high level of engraftment,and the oc/oc phenotype is completely rescued in a high percentage of these mice. Therefore,oc/oc pathology appears to be particularly sensitive to this form of early treatment of the ARO genetic disorder. View Publication

Fetal liver,the major site of hematopoiesis during embryonic development,acquires additional various metabolic functions near birth. Although liver development has been characterized biologically as consisting of several distinct steps,the molecular events accompanying this process are just beginning to be characterized. In this study,we have established a novel culture system of fetal murine hepatocytes and investigated factors required for development of hepatocytes. We found that oncostatin M (OSM),an interleukin-6 family cytokine,in combination with glucocorticoid,induced maturation of hepatocytes as evidenced by morphological changes that closely resemble more differentiated hepatocytes,expression of hepatic differentiation markers and intracellular glycogen accumulation. Consistent with these in vitro observations,livers from mice deficient for gp130,an OSM receptor subunit,display defects in maturation of hepatocytes. Interestingly,OSM is expressed in CD45(+) hematopoietic cells in the developing liver,whereas the OSM receptor is expressed predominantly in hepatocytes. These results suggest a paracrine mechanism of hepatogenesis; blood cells,transiently expanding in the fetal liver,produce OSM to promote development of hepatocytes in vivo. View Publication

Liver becomes the predominant site of hematopoiesis by 11.5 dpc (days after coitus) in the mouse and 15 gestational weeks in humans and stays so until the end of gestation. The reason the liver is the major hematopoietic site during fetal life is not clear. In this work,we tried to define which of the fetal liver microenvironmental cell populations would be associated with the development of hematopoiesis and found that a population of cells with mixed endodermal and mesodermal features corresponded to hematopoietic-supportive fetal liver stroma. Stromal cells generated from primary cultures or stromal lines from mouse or human fetal liver in the hematopoietic florid phase expressed both mesenchymal markers (vimentin,osteopontin,collagen I,alpha smooth muscle actin,thrombospondin-1,EDa fibronectin,calponin,Stro-1 antigens,myocyte-enhancer factor 2C) and epithelial (alpha-fetoprotein,cytokeratins 8 and 18,albumin,E-cadherin,hepatocyte nuclear factor 3 alpha) markers. Such a cell population fits with the description of cells in epithelial-to-mesenchymal transition (EMT),often observed during development,including that of the liver. The hematopoietic supportive capacity of EMT cells was lost after hepatocytic maturation,induced by oncostatin M in the cell line AFT024. EMT cells were observed in the fetal liver microenvironment during the hematopoietic phase but not in nonhematopoietic liver by the end of gestation and in the adult. EMT cells represent a novel stromal cell type that may be generated from hepatic endodermal or mesenchymal stem cells or even from circulating hematopoietic stem cells (HSCs) seeding the liver rudiment. View Publication